Monocytes and macrophages are derived from hematopoietic stem cells. Such stem cells are also precursors for other cells such as erythrocytes, granulocytes, platelets and lymphocytes. Once stem cells are committed to the monocyte/macrophage pathway, they differentiate into promonocytes. Promonocytes are small, round cells which exhibit extensive cell division and little to no adherence to tissue culture flasks. Promonocytes mature into monocytes which are slightly larger, exhibit less cell division and more adherence. Finally, monocytes mature into macrophages which are large cells which exhibit no cell division and strong adherence.
Continuous cell lines having monocyte/macrophage characteristics have a number of important uses. These cell lines can be used for characterization of normal monocyte/macrophage growth, maturation and function, for large-scale production of cytokines and mRNA for the production of recombinant DNA clones specifying these cytokines, for growth of viral and parasitic pathogens and, possibly, for production of vaccines for such viral and parasitic pathogens and for evaluation of drugs for such viral and parasitic pathogens.
Use of continuous cell lines having monocyte/macrophage characteristics has been limited by the difficulty in establishing such lines. Continuous cell lines have been isolated from both mouse and human sources, but the number of cell lines available for use is small, particularly those cell lines from human sources.
With few exceptions, human cell lines having monocyte/macrophage characteristics have been established only from leukemic patients. Thus, the cells of such cell lines are malignant and are less useful than cells of cell lines established from normal human cells for the study of normal human cell functions. In addition, cell lines established from malignant cells are less desirable than cell lines established from normal cells for the production of cytokines and vaccines since the malignant cells can secrete undesirable by-products which are difficult to separate from the final product. The two most commonly used human cell lines are U-937 [Sundstrom et al., Int. J. Cancer, 17:565-577 (1976)] and THP-1 [Tshuchlya et al., Int. J. Cancer, 26:171-176 (1980)]. Both cell lines were established by adapting leukemic cells to in vitro growth. Another human cell line occasionally used for production of monocyte/macrophage products is HL-60 [Collins et al., Nature, 270:347-349 (1977)]. This cell line appears to have been established from a less differentiated precursor cell since the cells, depending on which stimulating agent is used, can be stimulated to express some of the properties of a granulocyte or a monocyte/macrophage. HL-60 was also established from leukemic cells.
In the mouse system, approximately fifteen continuous cell lines having monocyte/macrophage characteristics are available. These cell lines were established from mice bearing tumors which occurred either spontaneously or, in most cases, were induced by treatment of mice with oncogenic viruses or chemical carcinogens.
The establishment of continuous cell lines having monocyte/macrophage characteristics from normal mice by the transfection of replication-defective oncogenic virus (SV40) DNA into peripheral blood monocytes has been described [Schwarzbaum et al., d. Immunol., 132:1158-1162 (1984)]. A similar technique has also been used to establish human cell lines [Nagata et al., Nature, 306:597-599 (1983)]. The human cell lines resulting from this technique are growth-factor dependent, requiring the addition of exogenous colony stimulating factor (CSF) for in vitro growth. Continuous cell lines requiring no exogenous growth factors have been established by introducing oncogenes from oncogenic viruses into monocytes [Pierce et al., 1986 Clinical Haematology (England) 15:573-596]. Cell lines containing oncogenes and viral DNA are not characteristic of normal cells and, therefore, are less desirable than cells lines established from normal cells for studying normal cell functions. In additon, these cell lines are less useful than cell lines established from normal cells for production of vaccines or cytokines because of harmful by-products which can be secreted by the oncogenes or virus particles.
Continuous cell lines having monocyte/macrophage characteristics have been established from some normal mammalian sources. Wardley et al.[Immunology, 39:67-73 (1980)]describe the establishment of continuous monocyte/macrophage cell lines from peripheral blood monocytes of a variety of species, including goat, pig, guinea-pig, sheep, rabbit, dog and cat. Very frequent, approximately daily, total medium changes were necessary to establish the cell lines. Attempts at establishing cell lines from human, horse and cattle sources were unsuccessful.
Salahuddin et al.[J. Exp. Med., 155:1842-1857 (1982)] describe the establishment of long-term (approximately five months) cell lines composed of replicating human monocytes/macrophages in suspension and adherent non-replicating macrophages. These cell lines were all established from peripheral blood of Epstein-Barr Virus (EBV)-seronegative individuals. Eventually, most of the cell lines terminated as non-replicating mature macrophages. However, two cell lines, which have grown in culture for several years, were established from peripheral blood mononuclear cells of one individual [Salahuddin et al., Biotechniques, 5(5):432-443 (1987)]. The method for preparing such cell lines required cell separation on supplemented discontinuous Percoil gradients, growth in media containing hydrocortisone and vitamin D.sub.3, weekly, total medium changes, gentle, continuous agitation, and peripheral blood from EBV-seronegative individuals. The method described by Salahuddin et al. appears to have worked only in two instances of approximately twenty-eight instances tried without any apparent explanation of the successes and failures or selection criteria for the starting cells other than that they be EBV-seronegative. In the absence of a reliable method for producing such cell lines the happenstance creation of just two cell lines is not enabling to those wishing to reproduce Salahuddin et al.'s work.
There exists a need for a method for establishing continuous cell lines in vitro having monocyte/macrophage characteristics from a wide variety of normal cell sources and mammalian species.